The object of the invention is novel cell lines so-called transcomplementation cell lines which enable the packaging of recombinant retroviral RNAs carrying nucleotide-sequences derived from genes which in some instances might be of therapeutic interest (called transgenes), aiming at transferring and expressing these transgenes in eucaryotic target cells. The invention also relates to expression vectors for the transcomplementation of defective retroviral vectors.
The transfer of genes for therapeutic purposes or somatic xe2x80x9cgene therapyxe2x80x9d consists in inserting a xe2x80x9crepairerxe2x80x9d gene (transgene) in the somatic cells of a constituted organism in order to compensate for the dysfunction of an endogenous gene, or even to add a novel function for a therapeutic purpose. The resulting genetic change is likely to be transmitted to the daughter cells of the manipulated cell but it will not be inherited. The normal counterpart of altered DNA sequences is thus transformed into a medicine.
Various approaches are currently being explored in order to introduce into target cells genes or more generally, nucleotide sequences, for therapeutic purposes. These target cells might be directly the cells benefiting from the therapeutic intervention or cell-intermediate between the vector carrying the transgene and the cells to be treated.
Vectors currently under use for gene transfer into target cells are derived either from inactivated viruses, like retroviruses or adenoviruses, or else from macromolecular conjugates. Retroviruses usually address a target tissue including a stem cell pool which can be manipulated ex vivo; whereas, when the target tissue is made of terminally differentiated cells or embedded into an organ where architectural constraints have major functional consequences, like the lung, gene transfer must be performed in vivo, by means of recombinant adenovirus for instance.
Gene therapy finds applications in diseases as diverse as hereditary disorders due to the alteration of a single gene, such as Duchenne""s myopathy, lyososomal diseases, cystic fibrosis or acquired disorders such as AIDS, cancers, thrombo-embolic disease or degenerative neurological diseases and inherited hematological diseases.
The development of retroviral vectors more efficacious than the existing tools constitute a major objective. In fact, retroviral vectors have demonstrated their efficacy in stably and permanently transferring genes where classically the cell-targets for the transfer undergo mitoses and ideally include a contingent of stem cells.
Current limitations stem from still inadequate infectivity of the viruses used and/or too moderate a level of transcription. Additional potential limitations deal with the safety of the transfer which to date remain unsatisfactory. This is both related to the risk for generation of replication-competent viruses and to the concomitant transmission of endogenous sequences which are generated by packaging cell-lines and may be packaged together with the defective recombinant virus genome.
The development of more efficacious and safer vector systems than those currently available is a matter of great significance.
Whenever the transgene to be expressed is delivered by means of a retroviral vector, the manufacture of stocks of retrovirus vector particles carrying along the transgene is a compulsory step preceding infection of target cells.
In order to achieve this, one should proceed with transcomplementation of the defective retrovirus vector carrying the transgene by the viral proteins, the corresponding encoding sequences of which being first deleted to be replaced by the transgene. This transcomplementation step can be performed passing through packaging cells. These cells are genetically engineered cells comprising the genetic material required for synthesis of viral components, in particular ENV envelope proteins, GAG nucleoproteins and POL reverse transcriptase, which allows for viral replication. The gag, pol and env genes are transfected into packaging cells through the transfection of the nucleic acid sequences which contain them, by means of a transcomplementation vector defective for the Psi-sequence which is otherwise mandatory to achieve virus packaging.
Expression of gag, pol and env genes within the packaging cells allows for further packaging of retroviral recombinant RNA sequences recombined with the transgene carried by a retroviral vector including the Psi-packaging sequences. Packaging may proceed following transcomplementation by the viral proteins encoded by the gag, pol and env sequences.
Currently available packaging cell lines are derived from cells of murine origin and harbour the following drawbacks:
1 Co-packaging of endogenous retroviral sequences (MCF, VL30, and even retrotransposons in some instances), together with defective constructs. Sequences which are co-packaged may as well integrate into the host genome following infection of gene transfer target cells.
2-Within these packaging cell lines, expression of complementing proteins is driven off a retrovirus LTR. Although third generation cell-lines make use of complementing retrovirus constructs including several mutation or deletion sites, maintaining LTR sequences might result in potential disadvantage; these sequences might well be able to induce genetic recombination with defective constructs to be complemented.
This has already been observed even from so-called third generation cell-lines where GAG and POL proteins encoding sequences, are being transfected separately from envelope coding sequences in order to reduce the likelihood for competent virus-generating combinations.
Envelope-encoding sequences currently under use to complement defective retroviruses towards gene transfer into mammalian cells and in particular of primate and human origin are amphotropic envelopes. It remains unsure that those cell-types that one wishes to infect towards gene therapy purpose would harbour enough receptors to amphotropic viruses.
All these elements justify efforts towards improvement in the safety of retrovirus-mediated gene transfer.
The object of the invention is to provide means to engineer novel packaging cell-lines to solve at least part of the abovementioned issues.
The cell-lines of the invention allow for the improvement of gene transfer safety since they are developed from cell-lines originating from animal species devoid of endogenous retroviral sequences identified to date and using, in a preferred embodiment non-viral promoters, to drive the expression of at least part of the viral complementing proteins, in particular inducible promoters for the conditional synthesis of defective vectors complementing proteins, especially of the envelope, upon request, whenever an infectious virus supernatant is to be prepared.
In addition GAG and POL proteins complementation is mediated by nucleotide sequences of a Friend retrovirus from which the vector has been built.
In order to implement the gag, and pol sequences are being transfected into the packaging-dedicated cell-line separately from envelope encoding sequences.
These cell-lines also lead to improvement in transfer efficiency through the use of envelope sequences, of human origin in particular, in order to circumvent the potential insufficient expression of the receptor to an amphotropic virus at the surface of human cell-targets for gene therapy; in particular potentially in case haematopoietic stein cells represent the targets.
According to a first embodiment, the object of the invention is an expression vector for the transcomplementation of a retroviral vector, which permits the transfer and/or integration within the genome of a target cell of a chosen nucleotide sequence (termed xe2x80x9ctransgenic sequencexe2x80x9d), characterized in that it comprises:
a) a nucleotide sequence termed env, coding for the polypeptides derived from the envelope (ENV polypeptides) of a retrovirus of the spumavirus family, the ENV polypeptides permitting the packaging of retroviral RNAs, and
b) transcription regulator signals controlling the expression of the env sequence, said expression vector being devoid of packaging signal sequences.
Retroviruses of the spumavirus (foamy virus) family are retroviruses one may be able to isolate from animal or human cell cultures.
A retrovirus named HSRV which belongs to the spumavirus family was isolated from a patient with nasopharyngeal carcinoma in 1971 (Hachong et al., J. Natl. Cancer Inst. 46, 299-307). The retrovirus HSRV has been cloned and sequenced and its sequence has been published by Flxc3xcgel R M et al (the EMBO Journal, vol. 6, nxc2x07, PP 2077-2084, 1987). This publication describes in particular the HSRV retrovirus envelope sequence.
Advantageously, the HSRV retrovirus envelope sequence used in the invention codes for a glycoprotein which can be recognized by a large number of surface receptors on human cells and in particular a glycoprotein which can make use of more receptors than would the amphotropic envelope glycoproteins, among a population of cells of human origin. According to an additional advantage of the invention, the envelope sequence under use harbour a tropism specific to human or primate for gene transfer in man or primate.
In particular, the HSRV-env sequence does interestingly harbour a tropism for human haematopoietic progenitor cells.
Besides haematopoietic stem cells, human primary cells such as fibroblasts or lymphocytes can be successfully targeted (lytic infection). Chronic infection by a defective virus has been evidenced in man within glandular epithelium or muscular tissues. Human originated cell-lines such as cells of epithelial or lymphocytic origin as well as HeLa can also represent appropriate targets. In rabbit, whatever the origin of primary cell tested, every one of them showed permissive to infection. The infection spectrum is thus very wide.
The expression of the env sequence being included in the transcomplementation expression vector can be driven off retrovirus transcription regulatory signals. In particular, these regulatory signals can be derived from retroviral LTR-sequence such as Friend retrovirus.
According to the present invention, the Friend virus strain under use is a strain identified as particularly virulent. The isolate 1-5 of the ecotropic Friend murine leukemia virus was obtained from long-term bone marrow cultures infected by the Friend virus complex which induces polycythemia (FV-P) (Mathieu-Mahul et al., 1982). The FB29 strain of F-MuLV derived from the isolate 1-5 (Sitbon et al., Cell, 47: 851-859, 1986) is responsible for cytolytic and leukemogenic effects on erythroid cells, leading to severe early hemolytic anemia followed by late erythroleukemia in susceptible mice inoculated at birth. The regions responsible for the erythroleukemia were localized in the U3 region of the viral LTR (Sitbon et al., 1986, Sitbon et al., PNAS USA, 88: 5932-5936, 1991). The principal determinant of the hemolytic anemia seems to depend on specific envelope sequences of the FB29 strain; its severity may be affected by three distinct regions, including a structural segment of the envelope, enhancer sequences of transcription localized in the U3 region and, finally, sequences of the U5-gag-pol regions (Sitbon et al., J. Virol. 64: 2135-2140, 1990). Furthermore, electron microscopical analyses of the viral particles have confirmed a significantly higher packaging capacity (1.5 to 2 log).
The spumavirus type retrovirus env sequence can thus be placed under the control of LTR sequence or of a part of the LTR sequence from the Friend virus FB29-strain sufficient to drive the transcription of the env-nucleotide sequence.
According to another embodiment of the invention, the expression vector for the transcomplementation is characterized in that the transcription regulator signals of the env sequence comprise a non-viral promoter. If the case arises, this promotor is followed either by the polyadenylation signal from SV40-virus or by another polyadenylation sequence.
The use of a non-viral promoter improves the safety characteristics of the thus engineered transcomplementation vector.
In addition, expression of the env-sequence can be triggered upon request, in case the promotor under use is of inducible type; i.e., the promotor can be activated by a molecule of choice, such as a pharmacologic compound in particular.
The gag and pol sequences which are being transfected separately can also be placed under the control of an inducible promoter.
Alternatively, transcription signals controlling the expression of the env- and/or expression of gag and pol-sequences can involve a conditional promotor the expression of which is restricted to defined cell-types.
One inducible type promotor suitable for the implementation of the invention is for instance, the promoter of the retinoic acid xcex2 receptor (RARxcex2 promoter).
This promoter has been described by De Thxc3xa9 et al., in Nature, vol. 49, nxc2x0 6254, pp 177-180 dated Jan. 11, 1990.
A preferred expression vector for transcomplementation is such that the spumavirus family retrovirus env sequence is placed under the control of an HindIII-BamHI fragment of the retinoic acid xcex2 receptor, as described in De Thxc3xa9""s et al., abovementioned publication.
According to another embodiment of the invention, the expression vector for transcomplementation with respect to the envelope is characterized in that it comprises:
a) a nucleotide sequence termed env sequence, coding for the polypeptides derived from an envelope, for example an amphotropic envelope, for example the envelope 4070A of Moloney leukaemia virus (Mo-MuLV), and
b) non-viral transcription regulator signals controlling the expression of the env sequence, containing, for example, an inducible or conditional promoter for example the RAR-xcex2 promoter.
According to a particular embodiment of the invention, the expression vector for transcomplementation is characterized in that the nucleotide sequence of the expression vector for transcomplementation, coding for the envelope polypeptides, is modified, for example by replacement of nucleotides by an arrangement of nucleotides coding for a polypeptide or glycoprotein recognized specifically by a defined cell type, or by the addition of such an arrangement.
According to another embodiment, the invention also provides for an expression vector for the transcomplementation of a retroviral vector, permitting the transfer and/or integration within the genome a target cell of a chosen nucleotide sequence (termed xe2x80x9ctransgenic sequencexe2x80x9d), including:
a) a nucleotide sequence termed gag coding for the polypeptides derived from a nucleoprotein (GAG polypeptides) of a retrovirus of the Friend retrovirus type,
b) a nucleotide sequence termed pol coding for the derived polypeptides including a reverse transcriptase protein and an integrase (POL polypeptides) of a retrovirus of the Friend retrovirus type,
c) non-viral transcription regulator signals controlling the expression of the gag and pol sequences, these signals containing, for example, an inducible or conditional promoter, for example the RAR-xcex2 promoter, and the abovementioned expression vector lacking encapsidation signals.
According to the invention, expression vectors for transcomplementation can be of an integrative type whether spontaneously or upon selection, or on the contrary, be episomal vectors.
The object of the present application also comprises a eukaryotic cell for the encapsidation of recombinant retroviral RNAs by transcomplementation, present on a retroviral vector which permits the transfer and the integration within the genome of a target cell of a chosen nucleotide sequence (transgenic sequence), the said cell being characterized that it exhibits the following properties:
it is selected from the cells of animal species lacking endogenous retroviruses, preferably chosen from foetal or embryonic cells, in particular those of dogs or rabbits,
it has a homogeneous morphology which is stable over time,
it is not of tumour origin,
it has the capacity to be selected in a minimum culture medium lacking CO2, without prior transformation, and
it has a rapid rate of multiplication.
According to the invention, the phrase xe2x80x98without prior transformationxe2x80x99 signifies that the cell does not benefit from the addition of oncogenic sequences, prior to its selection.
The cell morphology is homogeneous and stable with time, as long as it is not significantly altered over a time period of 6 months or more.
According to an attractive embodiment of the invention, a eukaryotic cell for the encapsidation of recombinant retroviral RNAs by transcomplementation, present on a retroviral vector permitting the transfer and integration within the genome of a target cell of a chosen nucleotide sequence (transgenic sequence), the said cell being selected from the cells of animal species lacking endogenous retroviruses, preferably chosen from foetal or embryonic cells, in particular those of dogs or rabbits, is obtainable by selection in accordance with the following method.
a) culturing of chosen eukaryotic cells on ISCOVE rich medium (GIBCO) containing, in addiction, 10% of foetal calf serum and 10% of horse serum,
b) passage of the cultured cells in DMEM medium containing 20% of foetal calf serum and culture of the cells for one month in a medium without CO2,
c) selection of the cells having a rapid rate of multiplication (accelerated kinetics),
d) passage of the selected cells in DMEM medium containing 10% of foetal calf serum,
e) passage of the cells recovered in step d) in DMEM medium containing 10% of newborn calf serum (HyClone),
f) recovery of the cells selected at the end of step e).
A eukaryotic cell can accordingly be the foetal dog cell designated DOGOM1 deposited at the CNCM on Nov. 30, 1994 under number 1-1496.
The dog foetal cell thusby obtained satisfies the following selection criteria; it lacks endogenous retroviruses; the adhesive characteristics of the cell are satisfactory; it has a rapid growth rate and its morphology is homogeneous and stable this dog foetal cell can also easily be transfected and is permissive to high range passagings (testing for intensive artificial passage-rate); it is capable of supporting a LTC-IC test (Long term culture initiating cells) as described in the experimental section.
From the above selected cells, genetically engineered cells permitting the encapsidation of retroviral RNAs can be prepared for instance by transfection in the above described cells or by their infection with the nucleotide sequences coding for the transcomplementation polypeptides or glycoproteins, the said sequences being supplied by means of at least two vectors or even at least three vectors. For example, on the one hand, a transcomplementation vector consisting either in a plasmid vector or a retroviral vector capable of expressing the env sequences encoding for the polypeptides derived from the envelope of a retrovirus of the spumavirus family can be used; and on the other hand, a vector, for instance ail expression plasmid-vector or a retroviral vector for the transcomplementation of the gag and pol nucleotide sequences of a retrovirus of the Friend virus type.
According to another embodiment of the invention the transcomplementing cells are xenogeneic, allogeneic or autologous cells which are likely to permit in vivo delivery of an infectious defective vector into a patient. In the latter case the transcomplementing cells may represent the cell-target for infection with the retroviral vector. For example, endothelial, muscle or stromal cells can be used.
The present application concerns a recombinant cell permitting the encapsidation of recombinant retroviral RNAs by transcomplementation (transcomplementing recombinant cell), characterized in that it is a cell replying to the above given definition and it is genetically engineered, for example, by transfection or infection with:
on the one hand, a first expression vector for the transcomplementation of a retroviral vector permitting the transfer and/or integration within the genome of a target cell of a chosen nucleotide sequence (termed xe2x80x9ctransgenic sequencexe2x80x9d), comprising the following transcomplementing sequences:
a) a nucleotide sequence termed gag sequence coding for the polypeptides derived from a nucleoprotein (GAG polypeptides) of a retrovirus of the Friend retrovirus type,
b) a nucleotide sequence termed pol sequence coding for the derived polypeptides including a reverse transcriptase and an integrase (POL polypeptides) of a retrovirus of the Friend retrovirus type, and
c) transcription regulator signals controlling the expression of the gag and pol sequences,
the abovementioned transcomplementation vector lacking packaging signals and,
on the other hand, a second expression vector for the complementation with respect to the env proteins comprising a nucleotide sequence coding for the envelope polypeptides and elements for regulation of the expression of this sequence.
According to the invention, a xe2x80x98virus of the Friend virus typexe2x80x99 is a virus the gag, pol, and env coding sequences of which have homologies with those of the Friend virus following alignment to achieve packaging of retroviral RNA as described in the present application.
According to a first embodiment of the invention, the nucleotide sequence encoding for one or several envelope polypeptides is derived from a retrovirus of the spumavirus family. It might for instance involve sequences derived from HSRV.
According to another embodiment of the invention, the nucleotide sequence coding for envelope glycoproteins is derived from the 4070A sequence of Mo-MuLV virus.
The various cell-lines are genetically engineered by means of sequential transfections along the principle of third generation packaging cell-lines; i.e., using fragmentation in two or even three or more, distinct parts of the sequences encoding GAG, POL and ENV complementing proteins and of their associated regulatory elements. This procedure reduces the potential for genetic recombination which could generate replication competent virus particles.
According to one embodiment of the invention, the packaging cell-lines of the invention are in other words, characterized in that they comprise:
a) a nucleotide sequence termed gag sequence coding for the polypeptides derived from a nucleoprotein (GAG polypeptides) of a retrovirus of the Friend retrovirus type,
b) a nucleotide sequence termed pol sequence coding for the polypeptides derived from a reverse transcriptase and from an integrase (POL polypeptides) of a retrovirus of the Friend retrovirus type,
c) transcription regulator signals controlling the expression of the gag and pol sequences,
d) a nucleotide sequence termed env sequence, coding for the polypeptides derived from the envelope (ENV polypeptides) of a retrovirus of the spumavirus family, the ENV polypeptides permitting the encapsidation of retroviral RNAs, and
e) transcription regulator signals controlling the expression of the env sequence.
The above described packaging cells are in addition intended to be transfected by a retroviral vector carrying the chosen desired transgenic sequence which should be expressed in a target cell upon infection with the supernatant of the packaging cells.
According to another embodiment, the object of the invention is a packaging cell characterized in that it comprises:
a) a nucleotide sequence termed gag sequence coding for the polypeptides derived from a nucleoprotein (GAG polypeptides) of a retrovirus of the Friend retrovirus type,
b) a nucleotide sequence termed pol sequence coding for the polypeptides derived from a reverse transcriptase and from in integrase (POL polypeptides) of a retrovirus of the Friend retrovirus type,
c) transcription regulator signals controlling the expression of the gag and pol sequences and,
d) a nucleotide sequence termed env coding for the polypeptides derived from an amphotropic envelope, for example the envelope 4070A of Moloney leukaemia virus (Mo-MuLV),
e) transcription regulator signals controlling the expression of the env sequence.
Advantageously, the packaging cell-lines of the invention lack helper virus and endogenous viruses, thus providing with improved safety conditions.
The GAG, POL, ENV polypeptides which are mentioned in this application either do correspond to all of the polypeptides and/or glycoproteins expressed by the virus nucleotide sequences gag, pol, env; or to a part of these polypeptides and/or glycoproteins; or to modified variants through a truncation process in particular, in case those permit achievement of the sought transcomplementation towards infection of the cell targets.
A particularly attractive cell according to the invention is characterized in that it is the cell DOGOM1 deposited at the CNCM under number I-1496, recombined on the one hand with the gag, and pol nucleotide sequences of Friend retrovirus strain FB29 under the control of the transcription signals contained in the LTR sequence of the Friend retrovirus strain FB29, and recombined on the other hand with the env nucleotide sequence of a retrovirus of the spumavirus type under the control of an inducible promoter, for example the RARxcex2 promoter.
Control of the sequence coding for the spumavirus envelope by means of an inducible promoter should prevent a potential lytic effect in transfected cells as a consequence of expression of spumavirus sequences.
Another attractive recombinant transcomplementing cell according to the invention is characterized in that the transcription signals included into the LTR-sequence of the Friend FB29-retrovirus which control the expression of gag and pol sequences are replaced by a non-viral promoter followed by SV40-virus polyadenylation signal or an alternative polyadenylation sequence.
The gag and pol sequences can thus be placed under the control of for example an inducible promoter such as RAR-xcex2.
According to another embodiment, the subject of the invention is recombinant transcomplementing cells for the packaging of retroviral RNA, characterized in that they comprise the abovementioned gag and pol sequences, under the control of an or else a conditional promoter; and an env sequence, for example amphotropic and for example derived from the 4070A Moloney virus env sequence under the control of an inducible or a conditional promoter as well.
According to a particular embodiment of the invention, the transcomplementing cell is characterized in that the nucleotide sequence of the expression vector for transcomplementation, coding for the envelope polypeptides, is modified, for example by replacement of nucleotides by an arrangement of nucleotides coding for a sequence of a polypeptide or glycoprotein recognized specifically by a defined cell type, or by the addition of such an arrangement.
The thus obtained recombinant cells are in addition, transfected with a retroviral vector for the expression and/or transfer and/or integration within the genome of a target cell of a chosen nucleotide sequence (transgenic sequence), the said retroviral vector being advantageously constructed starting from the FB29 strain of Friend retrovirus.
The retrovirus vector thus can be constructed in placing the transgenic sequence to be expressed under the control of the LTR sequence, while the gag-, pol-, env-encoding sequences being deleted at least in part; and the packaging sequence being included inside the vector construct.
A useful vector to be used for the transfection of the abovementioned transcomplementing cells is the vector pFOCH29 shown in FIG. 1 and deposited at the CNCM under No. I-1326 on Jun. 30, 1993. The transgenic sequence to be expressed into the target cells is inserted at a site which is non-essential for its replication.
Other retrovirus vectors can be used. In particular pFOCH29 derivatives will first be considered but if the case arises deleted at least in part amidst the LTR sequences and for example of the U3 domain of the LTR sequence.
Another vector is the vector pFOCH29 PL shown in FIG. 2.
According to the invention, any defective retroviral vector which can be efficiently pseudotyped passing through the abovedefined complementation cell-lines, can be used for transgene gene transfer purposes.
A transgenenic nucleotides sequence is a nucleotidic sequence which is not naturally part of the vector genomic sequences, and in particular of the sequences which control expression, cloning or gene transfer. It may be either a natural or a synthetic sequence, especially a hybrid sequence.
xe2x80x98Transfer of transgenic nucleotides sequencexe2x80x99 means integration of a sequence carried by the vector, within the genome, or in a satellite of the latter, of a target cell transduced with this vector. Such a transfer might result from a recombination, in particular homologous recombination.
The retroviral vector can therefore mediate the permanent expression of an exogenous nucleotide sequence through the modified, genome of a target cell; the exogenous sequence being selected according to its ability to undergo integration into the genome of the target cells.
The abovedefined defective vectors which carry the transgenic sequence or the plasmid-expression vectors which carry the gag, and pol sequences on the one hand and the env sequences on the other hand for complementation towards packaging, are introduced inside the packaging cell-line preferably by transfection or electroporation for example. This transfection can result in formation of viral particles which are suitable for achieving recombination following target cell transduction. This is performed towards cloning transfer or expression of the transgenic sequence included in the vector.
The sequence of therapeutic interest which can be introduced in target cells by means of the vectors and the packaging cell-lines of the invention are for example sequences which correspond to the normal equivalent of a non-functional gene in the context of a particular disorder; or else to an antisense sequence or to a dominant negative mutant of a particular gene; or to a sequence encoding for a functional inhibitor of a gene; or else to a reporter marker gene; or to a regulatory gene or a regulatory sequence of a particular gene; or to a gene which might confer a new function to target cells.
The invention does provide with appropriate means for gene therapy of cancer either through gene correction or improvement of strategies intended for the destruction of tumor cells. According to the first technique one can aim at correcting either inherited mutations in the context of inherited predisposition to cancer or signal transduction dysfunction, such as rasoncogene- and homologues-pathways; dysfunction resulting in oncogenes activation, dysfunction which results in tumor suppressor genes inhibition as well as correction of abnormalities which favour genetic instability or else which involve DNA-repair.
According to the second strategy, the invention can be advantageously operated in order to activate prodrugs such as making use of Herpes virus thymidine kinase gene which transforms either Ganciclovir or Acyclovir into cytotoxic drugs or else making use of the Cytosine deaminase gene which transforms a 5-fluorouracile precursor into an active drug; or else in order to induce or stimulate the immune system through genetic engineering of tumour cells by means of cytokines genes for example; or through engineering of antigen-presenting cells or their precursor (haematopoietic stem cells) or through immune effector cells-, T-cells-, B-cells-, LAK cells- or TlL cells engineering.
As for correction of genetic disorders, of anemias, the invention can be applied to the correction of inborn errors of metabolism, of haemoglobinopathies, such as thalassemias or drepanocytosis, of haemostasis and coagulation disorders, or else correction of inherited myelination disorders or myopathies.
The invention can also usefully be applied to either therapy or prevention of infectious disease such as AIDS; to cardiovascular diseases; to disorders of the lung, skin, liver and digestive tract, neuromuscular, central nervous system, pleura and peritoneum, etc.
According to another embodiment of the invention the transgenic sequence encodes for an antigen or a genetic determinant or an antiidiotype.
According to another embodiment of the invention the transgenic sequence encodes for an antibody, in particular a single chain antibody including especially variable sequences which are responsible for antigen recognition.
A vector containing such an antigenic determinant could potentially be used as a permanent or transient vaccine or if the case arises in the context of a therapeutic protocol, for example in order to elicit an immune response.
The tools of the invention are also suitable for performing vaccination of patients against pathogens or adventitious agents whether permanently or transiently.
The transfer might be performed through transduction of either cells, tissues, organs or organisms.
Suitable cell targets for implementing the invention are for example fibroblasts, endothelial cells, mesothelial and mesenchymal cells, skin cells among which keratinocytes, liver cells among which hepatocytes, muscle cells, accessory cells of the central nervous system, glial cells, oligodendrocytes, astrocytes etc. . . . ; epithelial cells among which urotheliuma, glandulary epithelia among which that from mammary gland, pulmonary epithelium, digestive tract epithelium as well as cell-lines whatever their origin.